Many human genetic diseases (about 15%) are caused by genetic mutations that, by interfering with the correct messenger RNA intracellular maturation, compromise the accurate subsequent protein biosynthesis and induce synthesis of non-functional proteins. Mostly, the point mutations accountable for splicing defects concern gene sequences that are critical for the recognition of the primary transcript by the machinery appointed for processing the same. The donor and acceptor sites located at the exon-intron boundaries, as well as gene-specific regulatory elements in exons or introns (Cartegni L et al., 2002; Pagani et al., 2004) are among the most significant sequences. As a consequence of these mutations, various molecular events, which most frequently concern the exclusion of one exon from the mature transcript, the so-called exon skipping, may be induced.
It has been known for a long time that molecular changes in the processing of messenger RNA, which involve, for instance, exon skipping, represent the main etiopathogenic mechanism of various human diseases, among which hemophilia B, cystic fibrosis, and spinal muscular atrophy and familial dysautonomia, which share the seriousness of their clinical courses. Different types of mutations can induce exon skipping, and specifically mutations in the donor site (or 5′ splicing site), mutations in the acceptor site (3′ splicing site), or exonic mutations. As examples of different types of mutations that induce exon skipping, following are described three models of human diseases.
The defect in the coagulation factor IX (FIX) accounts for the onset of hemophilia B, a disease accompanied by varying degrees of hemorrhagic manifestations, sometimes very serious and disabling. In some cases, the disease is caused by splicing defects. In particular, the exclusion of exon 5 from mRNA during the splicing process is caused both by mutations at position −2 within the exon 5 donor site of the factor IX gene (F9), and by mutations at positions −8 and −9 within the poly-pyrimidine sequence in the acceptor site.
The limitations of the current hemophilia B therapy, which is mainly based on the frequent infusion of recombinant exogenous FIX or of FIX directly derived from plasma, emphasize the need of developing alternative approaches that are characterized by a greater efficacy and a long-lasting effect.
Cystic fibrosis (CF) is the most frequent lethal congenital hereditary disease in the Caucasian population: one newborn out of 2500-2700 born-alive infants is affected by it.
The pathogenesis of this disease is secondary to an anomaly of a protein designated as CFTR (Cystic Fibrosis Transmembrane Conductance Regulator) localized in the apical membrane of epithelium cells and having the function of regulating the hydroelectrolytic exchanges.
As a consequence of CFTR modification, the transfer of salts through cell membranes is compromised, mainly causing a production of secretions that could be defined as “dehydrated”: a sweat very rich in sodium and chlorine and a dense and viscous mucus that tends to obstruct the ducts, compromising the function of various organs and systems. In the course of several studies, many modifications in the CFTR gene sequence were identified as associated with cystic fibrosis, which induce exon skipping. In particular, skipping of exon 12 is caused both by mutations localized within the splicing donor site of the exon itself, and by exonic mutations.
Spinal muscular atrophy (SMA, OMIM 253300, 253550, and 253400) is a recessive autosomal neuromuscular disease characterized by degeneration of spinal marrow alpha motoneurons, with an estimated prevalence of 1/10,000 born. SMA is associated with clinical syndromes that range from extremely serious, with critical muscle hypotonia and weakness since birth, to milder forms in which the onset occurs later during childhood or adolescence. To date, no treatment for this disease, which generally leads to death at an age that depends on the seriousness of the case history, has yet been identified.
In 95% of cases, the disease is caused by absence of the SMN1 gene. In the human genome, there is a gene homologous to SMN1 called SMN2. However, expression of SMN2 is impaired by a synonymous mutation in the exon which results in an aberrant maturation of the messenger RNA with consequent skipping of exon 7 and inactivation of the gene itself. Approaches designed to increase the number of exon 7-containing SMN2 transcripts would therefore allow to apply a compensation therapy for the absence of the SMN1 gene thanks to the correct expression of SMN2, with considerable implications for a potential effective treatment for SMA.
Familial dysautonomia (“FD”, Rilay-Day syndrome, OMIM 223900) is an autosomal recessive disorder that affects the sensory and autonomic nervous system. FD is a very common disease with a carrier frequency of 1 in 27 in the Ashkenazi Jewish population and of 1 in 18 in Ashkenazi Jews of Polish descent. This neuropathy is characterized by poor development and progressive degeneration of the sensory and autonomic nerves. FD patients show a large number of symptoms due to the loss of neuronal function, including gastrointestinal dysfunction, cardiovascular instability, recurrent pneumonia, decreased sensitivity to pain and temperature, vomiting crisis, and defective lacrimation. To date there is only palliative therapy.
FD is caused by mutations that affect the IKBKAP gene, inducing an aberrant processing of its pre-mRNA. This gene encodes the ikappaB kinase complex associated protein (IKAP), which has a molecular weight of 150 kDa. IKAP is also known as elongator protein 1 (ELP1) that is a component of the human Elongator complex, which is required for an efficient RNA Pol II transcriptional elongation. IKAP has also been associated with other cellular functions in addition to its role in transcription.
As of the present date, there are three known mutations related to FD: an intronic non-coding point mutation (IVS20+6 T>C, c.2204+6T>C, NM_003640.2) and two missense mutations (R696P and P914L). Among these three mutations, it has been found that more than 99% of FD patients are homozygous for the IVS20+6 T>C at the donor splice site of intron 20. This mutation causes exon 20 skipping leading to a frameshift that generates a premature termination codon (PTC) in exon 21 of IKBKAP mRNA. Interestingly, this mutation does not completely abolish the inclusion of exon 20, resulting in a minimal expression of the full-length IKAP protein. However, in neurons, this amount is not sufficient to support a physiological activity, leading to the pathological condition.
During the splicing process, the small nuclear RNAs (snRNAs) play a primary role as essential components of the spliceosome, the cell machinery appointed to mediate the entire mRNA maturation process. In particular, the small U1 RNA (U1snRNA), 164 ribonucleotides in length, is encoded by genes that occur in several copies within the human genome and represents the ribonucleic component of the nuclear particle U1snRNP. The U1snRNA molecules have a stem and loop tridimensional structure and within the 5′ region they include a single-stranded sequence, generally 9 nucleotides in length, capable of binding by complementary base pairing the splicing donor site on the pre-mRNA molecule (Horowitz et al., 1994). FIG. 1 shows a schematic representation of the wild-type U1snRNA structure. The sequence in the 5′ region capable of recognizing the splicing donor site is shown paired with the consensus sequence of the splicing donor site in the primary transcripts of eukaryotic genes. Such a sequence exhibits varying degrees of conservation and is located at the exon/intron junction. The recognition mediated by the U1snRNA 5′ region is critical for defining the exon/intron junctions on the primary transcript and for a correct assembly of the spliceosome complex.
The increasing number of human genetic diseases associated with pre-mRNA splicing defects, and the frequent seriousness of the clinical course of the same, stimulated in the last few years the research for therapeutic molecules aimed at correcting splicing defects at the molecular level.
The use of modified U1snRNA molecules capable of inducing in vitro the correct inclusion of the exon and restoring the correct splicing of coagulation factor VII mRNA in case of mutations located at the 5′ss site is described in Pinotti M et al. 2008 and Pinotti M et al., 2009. The illustrated mechanism is based on the recognition and binding of the modified U1snRNA directly onto the 5′ mutated splicing site. However, this method presents a certain degree of non-specificity of action of the therapeutic snRNA molecule towards the target gene, due to the relative conservation of the 5′ss sites and consequent risk of interfering with the maturation of transcripts generated from other functional wild-type genes. Moreover, it requires the use of a U1snRNA modified for each mutation in the 5′ss.
The present invention demonstrates that modified U1 snRNA molecules complementary to intron sequences downstream of the 5′ splicing site (and herein defined as Exon Specific U1s, ExSpeU1), are capable of restoring, during the splicing process, the exon inclusion which was impaired by different types of mutations. In three different human genetic disease models of therapeutic interest (spinal muscular atrophy, hemophilia, and cystic fibrosis), co-owned U.S. patent application Ser. No. 13/878,355, filed on Apr. 8, 2013, and the work of Fernandez Alanis et al., 2012 demonstrated that a single ExSpeU1 or a group of ExSpeU1s are able to induce the inclusion of the corresponding exon for each disease model. In the work of Dal Mas et al., 2015, it was also shown the effect of the use of three specific ExSpeU1s (sm2, sm17 and sm21) in cellular and animal models of spinal muscular atrophy. In U.S. Ser. No. 13/878,355, it was shown that a single ExSpeU1 or a group of ExSpeU1s correct the exon skipping caused by mutations in the donor site, mutations in the poly-pyrimidine tract of the acceptor site, and mutations in regulatory exon sequences. The correction effectiveness obtained with the ExSpeU1s is the same as that described in the prior art, but it would guarantee a greater selectivity of action on the target gene transcript of therapeutic interest. The ExSpeU1 approach allows use of a single modified U1-snRNA for correcting a panel of different genetic mutations that cause exon skipping. Accordingly, there remains a need in the art for a solution for familial dysautonomia that is caused by exon skipping.